Inhibition of specific naphthylamide hydrolyzing enzymes (leucine aminopeptidase) with detoxified snake serum



United States Patent 3,336,204 INHIBITION OF SPECIFIC NAPHTHYLAMIDE HY.

DROLYZING ENZYMES (LEUCINE AMINOPEP- TIDASE) WITH DETOXIFIED SNAKE SERUMVan B. Philpot, Jr., 161 State St., Framingham, Mass. 01701 No Drawing.Filed Sept. 8, 1964, Ser. No. 395,015 4 Claims. (Cl. 195-1035) Thepresent invention relates to inhibition or neutralization of proteolyticenzymes and more particularly to a means and method of inhibiting orneutralizing a proteolytic, naphythylamide hydrolyzing enzyme which ispresent in living organisms.

Much biological research is now concerned with the identification ofenzymes and their role in diseases of the body. Such research is oftencomplicated by difficulties in identifying and, in some cases, findingmethods of inhibiting or neutralizing the enzymes being studied. Oftenenzymes are identified by their reaction with certain diagnostic testagents. For example, it is known that certain proteolytic enzymeshydrolyze naphthylamide and such enzymes may at least be partiallyidentified by reaction with naphthylamide. In certain cases the presenceor absence of particular enzymes can indicate the presence or absence ofdiseases of the body.

Accordingly it is an important object of this invention to provide ameans and method for inhibiting or neutralizing certain proteolyticenzymes.

It is another important object of this invention to provide a method inaccordance with the preceding object which permits at least partialidentification of such inhibited enzymes and which method is useful incharacterizing and identifying enzymes both in research and clinicaldiagnosis.

It has been found that certain naphthylamide hydrolyzing enzymes arepresent in the stomach tissue of humans who have gastric ulcers.Naphthylamide hydrolyzing enzymes are reported to be proteolytic and arereferred to as leucine aminopeptidase in the New England Journal ofMedicine at vol. 259, No. 10, page 469 (1958). The presence of suchenzymes which arepresent at the ulcer site can ordinarily be determinedby staining cryostat sections of an ulcerated stomach with a mixture ofa naphthylamide and diazotized-o-aminoazotoluene. When such sections aremade, the tissue surrounding the ulcer of the stomach wall stains anintense reddish violet color indicating the presence of proteolytic,hydrolytic enzymes. This intense reddish violet color does not resultwhen tissues spaced from the ulcer site are stained. It is believed thatthe enzyme or enzymes which cause the reddish violet color upon stainingare an etiological factor in the production of ulcers.

According to the method of this invention, the proteolytic naphthylamidehydrolyzing enzyme or enzymes causing the reddish violet color whenstained, can be inhibited or neutralized by treating with detoxifiedsnake serum. This method is useful as a research tool in understandingthe mechanism of gastric ulcers and in characterizing and diagnosing theenzymes surrounding a gastric ulcer and perhaps others present in otherdiseased tissue. Thus, certain enzymes can be characterized as reactingwith detoxified snake serum so that they do not give a red violet colorwhen stained with a naphthylamide.

Preferably the snake serum used is detoxified by heating to form aprecipitate therein and then removing the precipitate so that the snakeserum will not destroy living human tissue. In the preferred method ofthis invention, a proteolytic, naphthylamide hydrolyzing enzyme presentat the site of the human gastric ulcers in tissue closely surroundingsuch ulcers, is inhibited or neutralized by treating an enzyme specimenwith detoxified snake serum to cause a reaction between the enzyme andthe snake serum. The reaction is preferably carried out at a temperatureof from 20 to 37 C. for 15 min. to 1 hr. and causes the tissuecontaining the enzyme to show no red violet color change when stainedwith a naphthylamide.

The snake serum useful in the method of this invention is detoxified tothe extent that it is nontoxic to living organisms and can even beinjected into the body without causing tissue destruction and necrosis.Preferably detoxification is carried out by heating snake serum in testtubes in a water bath at 56 C. for 30 minutes. Following the heatingstep, a precipitation is formed within the snake serum which isdispersed in the serum and gives a milky appearance. This precipitate,which is capable of causing hemolysis in living organisms, is separatedand removed from the treated serum preferably by filtering orcentrifugation and decantation of the resultant clear, supernatanttreated serum. The partic ular temperature at which the serum is heatedmay vary as may the time of heating so long as toxic materials in theserum are destroyed without destroying that portion of the serum whichreacts with proteolytic naphthylamide hydrolyzing enzymes. Generally,heating occurs at temperatures of from about 45 C. to about 65 C. forfrom 'about five to about minutes. The time and temperature may varyinversely with each other. In some cases temperatures above 65 C. may beused for shorter time periods. Conversely, temperatures below 45 C. maybe used for longer time periods.

Rattlesnake serum is preferred for use in the method of this inventionalthough other snake serums may be used and obtained from widely varyingtypes of snakes including the Florida rattlesnake (Crotalus adamanteus),

water moccasin (Agkistrodoiz piscivorus), Japanese habu (Elaphequadrivirgata) and king snake (Lampropeltis getulus). The serum can beobtained in a conventional manner by cutting off a portion of the snakestail and allowing the blood'to flow into a sterile container. The serumis separated from the cells by centrifugation. The particularnaphthylamide indicator or test reagent used is preferablyL-leucyl-B-naphthylamide in admixture with diazotized-o-aminoazotoluene.The. L-leucyl-B-naphthylamide is a clear colorless liquid which can behydrolyzed to a red or red violet color in the presence of certainproteolytic, hydrolytic enzymes or naphthylamide hydrolyzing enzymessuch as leucine aminopetidases which are reported to be proteases. Thediazotized-oaminoazotoluene serves to deepen and intensify the colorchange brought about by hydrolysis of the naphthylamide. In a preferredindicator solution of this invention, 30 milligrams ofdiazotized-o-aminoazotoluene along with 1 cc. of a 1% solution ofL-leucyl-B-naphthylamide are mixed with 40 cc. of water and 10 cc. trisbuffer pH 7.1. In a specific example of this invention illustrating theinhibition or neutralization of certain proteolytic naphthylamidehydrolyzing enzymes by snake serum in accordance with this invention, abiopsy was performed on a living human and a stomach wall sample wasobtained which was immediately frozen. Multiple cryostat sections weremade of the stomach wall portion using a microtone and conventionalprocedures. The test tissue sections were placed on sample slides withtwo slides being made of a portion of each of the antrum, fundus andmidportion of the stomach wall. The tissue on the two slides of themidportion of the stomach were adjacent the site of a gastric ulcer inthe patent upon whom the biopsy was performed. Each of the slides wasair dried and one slide from each section of the antrum, fundus andmidsection, respectively, was stained with the preferred naphthylamideindicator solution described previously by immersing the slide for 4hours at room temperature in a Copland jar filled with the stainingsolution. It was observed that the midportion section showed a veryintense red violet color when viewed under the microscope. The tissuesections from the antrum and fundus not near the ulcer site did not showan intense red violet color but merely a red color. It is theorized thatcertain naphthylamide hydrolyzing enzymes are present in all sections,but, a specific naphthylamide hydrolyzing enzyme was present in thetissue surrounding the ulcer site which gave an intense red violet colorchange rather than merely a red color change with the naphthylamide. Theenzyme or enzymes which gave the intense red violet color change aretheorized to be etiological factors in producing ulcers.

A second set of slides carrying tissue sections from the antrum, fundusand midportion was treated by appyling a few drops of detoxified snakeserum to the tissue sections and incubating the slides for one hour at37 C. The snake serum was rattlesnake serum which was heated in a testtube in a water bath at 56 C. for 30 minutes followed by centrifugationof the resulting precipitate and separation of the detoxified serum.After one hour of incubation, the slides were washed in gently runningtap water and stained with the preferred naphthylamide solution as inthe first series. Surprisingly it was found that a particular enzymeorenzymes which cause the deep red violet color in the stomach tissuesurrounding the ulcer site was inhibited or neutralized by the snakeserum since no red violet color was observed in the tissue section fromthe midportion in this procedure. In addition, the tissue sections fromthe antrum and fundus showed a red color as previously found in thefirst series.

When similar sets of slides are prepared from the same stomach specimenand treated with saline and human serum in place of snake serum, inaccordance with the procedure followed for the second set of slides, theresultant stained color of each tissue section is substantially the sameas obtained with the first series of slides. In some cases the humanserum appears to intensify the red violet color in tissue specimens.

While a specific example of this invention has been described it will beobvious to those skilled in the art that many variations thereof arepossible. For example, the specific incubation period for the test serumwith the tissue specimen containing the proteolytic naphthlamidehydrolyzing enzyme can be varied. Similarly in some cases the enzyme maybe reacted with the detoxified snake serum by mixture in a test tubewith the enzyme in pure form removed from a tissue specimen. The snakeserum may be used in admixture with other enzymes or tissues containingsuch enzymes and then tested for a color change with naphthylamide todetermine whether or not such enzymes are similar toor give similarreactions with snake serum as does the particular enzyme or enzymessurrounding a gastic ulcer site. The snake serum may be useful toinhibit or neutralize the proteolytic, naphthylamide hydrolyzing enzymein other environments, and even therapeutically in living organisms.Other indicators can be used to test the presence or absence of areaction between snake serum and an enzyme tested.

In view of the many variations possible, this invention is to be limitedonly by the spirit and scope of the appended claims.

What is claimed is:

1. A method of characterizing a proteolytic, naphthylamide hydrolyzingenzyme comprising detoxifying snake serum by heating said snake serum toa temperature in the range of from about 45 C. to C.,

applying said snake serum to said enzyme to form mixture therewith,

introducing a naphthylamide indication solution into said mixture, andobserving the presence or absence of a red violet color with thepresence of a red violet color indicating no inhibition of said enzymeand the absence of said red violet color indicating inhibition of saidenzyme.

2. A method in accordance with the method of claim 1 wherein said snakeserum is rattlesnake serum.

3. A method in accordance with the method of claim 1 wherein said snakeserum is applied to said enzyme and incubated for at least 15 minutes ata temperature of at least 20 C.

4. A method in accordance with the method of claim 1 wherein saidnaphthylamide is L-leucyl-B-naphthylamide and hasdiazotized-o-aminoazotoluene incorporated therewith.

References Cited Chemical Abstracts, 5th Decennial Index, vol. 41-50(1947-1956), Subject Index TH-Z, pp. 132298 to 132305, published 1962.

Rutenbur-g, A.M., et al., The New England Journal of Medicine, vol. 259,N. 10, pp. 469-472 (1958).

A. LOUIS MONACELL, Primary Examiner.

HYMAN LORD, Examiner.

L. M. SHAPIRO, Assistant Examiner.

1.A METHOD OF CHARACTERIZING A PROTEOLYTIC, NAPHTHYLAMIDE HYDROLYZINGENZYME COMPRISING DETOXIFYING SNAKE SERUM BY HEATING SAID SNAKE SERUM TOA TEMPERATURE IN THE RANGE OF FROM ABOUT 45*C. TO 65*C., APPLYING SAIDSNAKE SERUM TO SAID ENZYME TO FORM A MIXTURE THEREWITH, INTRODUCING ANAPHTHYLAMIDE INDICATION SOLUTION INTO SAID MIXTURE AND OBSERVING THEPRESENCE OR ABSENCE OF A RED VIOLET COLOR WITH THE PRESENCE OF A REDVIOLET COLOR INDICATING NO INHIBITION OF SAID ENZYME AND THE ABSENCE OFSAID RED VIOLET COLOR INDICATING INHIBITION OF SAID ENZYME.